Inhibition of the gastric H+,K+-ATPase by plectrinone A, a diterpenoidisolated from Plectranthus barbatus Andrews
Carla Schultz a, Myllene P. Bossolani a, Luce M.B. Torres b, Maria Teresa R. Lima-Landman a,Antonio J. Lapa a, Caden Souccar a,∗
a Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Department of Pharmacology,Natural Products Section, 04044-020 Rua Tres de Maio 100, Sao Paulo, SP, Brazil
b Botanical Institute, Botanical Garden of Sao Paulo, SP, Brazil
Received 3 August 2006; accepted 22 September 2006Available online 11 November 2006
bstract
This work assessed the mechanism underlying the antisecretory gastric acid effect of Plectranthus barbatus Andrews (Lamiaceae) and activeonstituents. Popularly known as “false-boldo”, this plant is used in Brazilian folk medicine to treat gastrointestinal and hepatic ailments. Thelant aqueous extract (AE) and isolated compounds were assayed in vivo in pylorus-ligated mice, and in vitro on acid secretion measured as
14C]-aminopyrine ([14C]-AP) accumulation in rabbit gastric glands and gastric H+,K+-ATPase preparations. Injected into the duodenal lumen,he AE of the plant leaves (0.5 and 1.0 g/kg) decreased the volume (62 and 76%) and total acidity (23 and 50%) of gastric acid secretion inylorus-ligated mice. Bioguided purification of the AE yielded an active fraction (IC50 = 24 �g/ml) that inhibited acid secretion in rabbit gastriclands with a potency 10 to 18 times greater than that of the originating extract, on both the basal and stimulated acid secretion by histamine
+ +
His) (1 �M) or bethanechol (100 �M). At the same concentrations the gastric H ,K -ATPase activity was also inhibited. The active constituentas chemically identified as the abietanoid dienedione plectrinone A which reduced the H+,K+-ATPase activity with IC50 = 171 �M. The results
ndicate that inhibition of the gastric proton pump by this diterpenoid may account for the antisecretory acid effect and reputed antiulcer activityf Plectranthus barbatus.
2006 Elsevier Ireland Ltd. All rights reserved.
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eywords: Plectranthus barbatus; Lamiaceae; Acid secretion; Rabbit gastric g
. Introduction
Plectranthus barbatus Andrews (Lamiaceae), previouslynown as Coleus barbatus (Andr.) Bentham, is a species nativeo India and Tropical Africa, but well adapted in the Brazil-an Northeast and Southeast regions. A diversity of medicinalse of Plectranthus barbatus has been reported such as in
astrointestinal disorders, skin affections, pain, respiratory andenito-urinary diseases (Lukhoba et al., 2006). Organic extractsf different parts of Plectranthus barbatus were reported to
resent anti-inflammatory, antifungal, antibacterial (Matu andan Staden, 2003), antioxidant (Tamasiro et al., 1998) andytotoxic (Costa and Nascimento, 2003) activities. Hypoten-ion and bradycardia were reported after intravenous injectionf the dichlorometane extract in anesthetized rats (Kelecom,983).
Because the bitter taste of Plectranthus barbatus leavesesembles that of the Chilean Peumus boldus, a Monimiaceaenown for its hepatoprotective effects (Speisky and Cassels,994), species grown in Brazil are known as false-boldo orrazilian-boldo, and used in medicinal teas to treat gastrohep-tic disorders and intestinal disfunction (Lorenzi and Matos,002). Confirming these medicinal effects, we have previouslyhown that the aqueous extract (AE) of Plectranthus barbatus
nhanced the intestinal transit in mice, protected againstastric lesions induced by ethanol or cold-restraint stress, andeduced the total acidity of gastric secretion in pylorus-ligatedats (Fischman et al., 1991; Lapa et al., 1992). The plant
epatoprotective effect was reported in rats with obstructiveholestasis (Battochio et al., 2005).
Extensive chemical studies on Plectranthus species showedhe presence of diterpenoids, phenolic compounds and essentialils (Abdel-Mogib et al., 2002; Kerntopf et al., 2002; Sousa etl., 2004). The leaves of Plectranthus barbatus grown in Brazilre rich in abietane diterpenes, whereas the roots of the Indianpecies contain mainly labdane diterpenes such as coleonolnd the activator of adenylyl cyclase forskolin (Zelnik et al.,977). Although referred in the literature as synonym of Coleusorskohlii (Willd.) Briq., the Brazilian specimen does not containorskolin in detectable amounts (Sousa et al., 2004).
Despite the widespread medicinal use of Plectranthus barba-us and diversity of isolated compounds, the active constituentsccounting for the plant antacid/antiulcer activity and theirechanism of action are still unknown. These studies are
equired for standardization and registration of plant-derivedhytomedicines by the local Regulatory Agency.
This work aimed to assess the mechanisms involved inhe antisecretory gastric acid activity of Plectranthus barbatusxtract and its main active constituent plectrinone A (Fig. 1), anbietane diterpene previously reported in the leaves of Plectran-hus barbatus (Ruedi, 1986). Using a bioguided fractionation ofhe plant AE, this diterpenoid was isolated and its effect was eval-ated comparatively to that of all intermediate fractions obtainedimultaneously from the original extract.
. Materials and methods
.1. Plant extracts, fractionation and identification oflectrinone A
Plectranthus barbatus was cultivated at the Pluridisciplinaryenter of Chemical, Biological and Agronomic Studies of theniversity of Campinas in the State of Sao Paulo, under the
upervision of Drs. P.M. Magalhaes and I. Montanari. A voucherpecimen was deposited at the Herbarium of that Institution asPQBA-UNICAMP 271.
The ground dried leaves of Plectranthus barbatus were
xtracted (5%, w/v) with hot water (72 ◦C, 30 min), theE was concentrated under vacuum and freeze-dried (yield:9.5%, w/v). The dried powder of AE (50 g) was parti-ioned with water (500 ml) and ethyl acetate (EtOAc: 3×,
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harmacology 111 (2007) 1–7
00 ml) yielding the organic (OF, 1.8%) and aqueous (AF,7.5%) fractions. Plectrinone A was purified from OF usingephadex LH-20 (Pharmacia Biotech) column chromatogra-hy eluted with methanol (MeOH), followed by silica-gelolumn chromatography of the active fractions eluted with hex-ne/EtOAc (1:1). Fractionation was monitored by thin-layerhromatography on silica gel plates (G254, Merck) eluted withtOAc/MeOH/H2O (100:13.5:10) and revealed with UV detec-
ion (λ = 254 + 356 nm) or sulfuric-acid vanillin. The purificationrocess was monitored by in vitro testing of the antisecretoryctivity in isolated rabbit gastric glands. All intermediate frac-ions were tested. Plectrinone A was identified by 1H and 13CMR spectral analysis, and assignments of the 1H and 13C NMR
ignals were based on HMQC and HMBC experiments.
.2. Animals
Male Swiss albino or F1 mice (25–30 g) (Vela et al., 1997)ere used. All animals were housed under a controlled dark/light
ycle and temperature (22 ± 2 ◦C), with free access to stan-ard chow diet and tap water. When necessary, animals wereeprived from food allowing free access to water 15–18 h beforehe experiment. The experiments were conducted in accordanceith the Guide for the Care and Use of Laboratory Animals of
he USA NIH and were approved by the institutional Animalnvestigation Ethical Committee (CEP 307/00).
.3. Gastric acid secretion
Pylorus ligature was performed in fasting mice under lightther anesthesia as previously described (Vela et al., 1997).he test extract and fractions were injected into the duodenal
umen and the abdominal wall was sutured. After 4 h the ani-als were killed by ethyl ether inhalation, a second ligature was
laced around the esophago-cardiac junction and the stomachas removed. The gastric secretion was collected and its finalolume and pH were determined after washing the mucosalide of the stomach with 2 ml of distilled water. Total acidityf the gastric juice was titrated with 0.01 N NaOH, using 2%henolphthalein as indicator.
.4. Gastric glands isolation
White rabbits (2–3 kg) were killed with an overdose of pen-obarbital sodium, i.v., the abdomen was cut open, the stomachas perfused through the aorta with phosphate buffered saline
PBS, in mM: NaCl 149.6, K2HPO4 3, NaH2PO4 0.64, pH 7.4t 37 ◦C), and excised. The fundus and antral regions wereiscarded, the mucosa was freed from the muscular layers,inced and digested in a collagenase solution (Sigma type IA,
.2 mg/ml) continuously gassed with 95% O2 and 5% CO2,or 30 min at 37 ◦C. The gastric glands suspension was filteredhrough a nylon mesh (750 nm), rinsed four times with PBS
ontaining 0.5% bovine serum albumin by sedimentation, andesuspended in PBS at a final concentration of 30 mg/ml. Theiability of the isolated gastric glands was greater than 90% asetermined by trypan-blue dye exclusion.
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.5. [14C]-aminopyrine accumulation
Accumulation of the weak base [14C]-aminopyrine ([14C]-P) was used as a measure of acid secretion by the gastric glands
Berglindh and Obrink, 1976). Samples of the glands suspen-ion (15 mg dry weight in 1.5 ml final volume) were incubatedn triplicates with either the vehicle, the test fractions or the ref-rence drugs ranitidine (10 �M) and atropine (10 �M), at 37 ◦Cs previously described (Lapa et al., 2000). After 15 min, 75 nCif [14C]-AP and the agonists histamine (1 �M) or bethanechol100 �M) were incubated for further 15 min. At these concentra-ions both secretagogues induced maximal stimulatory effect asetermined in preliminary concentration-response relationships1–100 �M). Nonspecific trapping of [14C]-AP was determinedn samples containing 0.1 mM 2,4-dinitrophenol. The reactionas stopped by adding 1.5 ml ice-cold PBS, the pellets wereashed briefly twice with PBS at 4 ◦C, and digested with50 �l nitric acid at 60 ◦C for 10 min. The radioactivity of theupernatants and digested pellets was counted on a scintillationeta-counter (Packard Instruments).
.6. Preparation of gastric H+,K+-ATPase
Rabbit gastric membrane vesicles enriched in H+,K+-ATPaseere prepared by a sucrose discontinuous density gradient
entrifugation (Kubo et al., 1995). Mucosal scrapings fromabbit stomachs were minced and homogenized in ice-cold0 mM Tris–HCl buffer, pH 7.4 using a glass homogenizer20 up/down strokes). The homogenate was centrifuged at3,500 rpm for 20 min, the supernatant was collected andentrifuged at 30,000 rpm for 1 h. The resulting pellet was resus-ended in sucrose 2.5 mM, layered over a 30% sucrose solutionnd centrifuged at 24,000 rpm for 2 h. All steps of the prepara-ion were carried out at 0–4 ◦C. Membrane vesicles containing
+,K+-ATPase were washed free from sucrose and stored at70 ◦C. Protein content was determined by the method of
Bradford, 1976), using bovine serum albumin as the standard.
.7. Assay of H+,K+-ATPase
ATPase activity of gastric membrane vesicles was determinedy measuring the inorganic phosphate released after hydrolysisf ATP (Fiske and Subbarow, 1925). Samples containing 40 mMris–HCl buffer, pH 7.4, 2 mM MgCl2, 10 �g membrane proteinith 1 mM ATP, and 20 mM KCl were incubated in triplicates,
or 20 min at 37 ◦C in the absence and presence of the testedompounds. The reaction was terminated by addition of 1 mlce-cold trichloroacetic acid 10%. Enzymatic assays with sam-les containing 0.1 mM ouabain were performed to exclude theresence of Na+,K+-ATPase. Samples incubated with the pro-on pump inhibitor omeprazole in acidified assay buffer (pH 5.5)ere used for positive control.
.8. Drugs and chemicals
Drugs used were: ATP dissodium salt, atropine sulfate,ethanechol chloride, histamine dihydrochloride, collagenase
dimethylamine-14C]-aminopyrine (106 mCi/mmol, Amershamnt.). All other chemicals were of analytical grade. The test frac-ions were dissolved in 3% tween 80, for in vivo administration,nd in 0.2% DMSO for the in vitro experiments. Appropriateoncentrations of the vehicle prepared with PBS were used asontrol.
.9. Statistics
Data were expressed as mean ± S.E. Half-maximal inhibitoryoncentrations (IC50) and 95% confidence intervals (CI) wereetermined from inhibition curves using the software Graph-ad Prism version 4.00 for Windows (GraphPad Software, Saniego, California, USA). The results were compared using one-ay analysis of variance (ANOVA) followed by the Dunnet
est. Differences among data were considered significant at< 0.05.
. Results
.1. Effects of the precursor extracts on acid secretion inivo
Intraduodenal injection (i.d.) of the AE of Plectranthus bar-atus (0.5 and 1.0 g/kg) to pylorus-ligated mice decreased theolume (by 62 and 76%) and total acidity (by 23 and 50%,espectively) of gastric secretion compared to control values,fter 4 h (Table 1). The pH of the collected secretion was nothanged. Comparatively, treatment of pylorus-ligated mice withhe antagonist of histamine H2 receptors, ranitidine (50 mg/kg,.d.), reduced the volume and total acidity by 76 and 80% ofontrol, respectively, while the pH was increased by 1.1 unitTable 1).
The aqueous fraction resulting from AE partition did not sig-ificantly change the parameters of acid secretion, while the OF0.1 g/kg, i.d.) reduced the volume and total acidity of gastricecretion by 71 and 77%, respectively, and increased the pH by.2 unit, compared to the vehicle-treated group (Table 1).
.2. Effects on acid secretion in vitro
Incubation of fraction OF (0.1 to 1.0 mg/ml) in isolated gas-ric glands samples produced a concentration-related decreasef acid secretion measured as uptake of [14C]-aminopyrine. Theehicle alone (DMSO 0.2%) did not significantly change theasal [14C]-AP accumulation or that maximally stimulated by�M histamine (235.1 ± 29.1% of basal) or 100 �M bethane-hol (171.5 ± 17.5% of basal) (Fig. 2). In presence of 0.3 and.0 mg/ml of fraction OF, the basal [14C]-AP accumulation was
ecreased by 62 and 73%, respectively, of those values deter-ined in presence of the vehicle alone. Low concentrations of
raction OF (≤0.1 mg/ml) were ineffective on the basal acidecretion (Fig. 2a).
4 C. Schultz et al. / Journal of Ethnopharmacology 111 (2007) 1–7
Table 1Effects of Plectranthus barbatus aqueous extract (AE), aqueous fraction (AF), and organic fraction (OF) administered intraduodenally (i.d.), on gastric acid secretionin 4 h pylorus-ligated mice
Treatment Volume (ml) pH Total acidity (mEq [H+]/l/4 h)
Results are mean ± S.E.; n = 6 for all groups, AE and AF were prepared in saline (coa Different from control group (ANOVA, p < 0.05).b Different from the vehicle-treated group (ANOVA, p < 0.05).
Fig. 2. Effect of the organic fraction (OF, 0.1 to 1.0 mg/ml) isolated from theaqueous extract of Plectranthus barbatus, the vehicle (V), ranitidine (Ran,10 �M) and atropine (ATR, 10 �M) on: (a) the basal [14C]-aminopyrine accu-mulation, and after stimulation by (b) histamine (His) (1 �M) or (c) bethanechol(100 �M) in isolated rabbit gastric glands. The columns and vertical bars aremean ± S.D. of three experiments in triplicates. (*): Different from the basal(p < 0.05).
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At 0.1 to 1.0 mg/ml OF also reduced [14C]-AP accumulationtimulated by histamine (His, 1 �M), by 36 to 88%, as wells that induced by bethanechol (�Ch, 100 �M), by 45 to 77%,f control values (Figs. 2b and c). Ranitidine (10 �M) and thentimuscarinic agent, atropine (10 �M), used as reference drugs,ecreased [14C]-AP accumulation in glands incubated with theame concentrations of His and �Ch by 64 and 43% of control,espectively (Figs. 2b and c).
OF fractionation in a LH-20 column yielded five fractions,wo of them (OF2 and OF3, 0.05 to 0.2 mg/ml) reduced the basal23 to 84%) and stimulated [14C]-AP accumulation by His (24 to5%) or �Ch (56 to 86%). The remaining fractions were inactivep to 0.2 mg/ml. Both active fractions were then combined intonew active organic fraction (OFa).
Subsequent purification of OFa in silica-gel column chro-atography yielded six subfractions that were assayed in rabbit
astric glands. The most active subfraction (OFa2, 1 to 30 �g/ml)ecreased the basal [14C]-AP accumulation (by 26 to 69%)nd reduced [14C]-AP accumulation induced by 1 �M His (14o 74%) or 100 �M �Ch (19 to 65%) (Fig. 3). Compared tohe originating organic extract, the antisecretory effect of theurified fraction was 18 times greater on the basal acid secre-ion (IC50 = 24 �g/ml), and 11 and 7 times more effective on14C]-AP accumulation stimulated by His (IC50 = 12 �g/ml) andCh (IC50 = 13 �g/ml), respectively. This fraction containedlectrinone A (C20H22O6) (Fig. 1) as the majority (>80%)ompound.
.3. Effects of plectrinone A on the gastric H+,K+-ATPase
Inhibition of the basal acid secretion as well as that stimu-ated by both secretagogues in presence of the purified fractionsndicated inhibition of the proton pump, the final commonathway of acid secretion. In control preparations of rabbitastric membrane vesicles, the H+,K+-ATPase activity deter-ined in presence of 1 mM ATP was 37.1 ± 0.34 �mol/Pi/mg
rotein/h (n = 15). Incubation of plectrinone A (1 �M–1 mM)ecreased the H+,K+-ATPase activity proportionately to the con-
entration with IC50 = 171 �M (CI: 0.78–375 �M) (Fig. 4). Athe same experimental conditions the proton pump inhibitormeprazole (10 nM to 1 mM) inhibited the H+,K+-ATPase withC50 = 12.4 �M.
C. Schultz et al. / Journal of Ethnopharmacology 111 (2007) 1–7 5
Fig. 3. Effect of the purified fraction OFa2 (1 to 30 �g/ml) isolated from theaqueous extract of Plectranthus barbatus, the vehicle (V), ranitidine (Ran,10 �M) and atropine (ATR, 10 �M) on: (a) the basal [14C]-aminopyrine accu-mulation, and after stimulation by (b) histamine (1 �M), or (c) bethanechol(m(
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p < 0.05).
. Discussion
The presented data show that plectrinone A is the most activesolated constituent involved in the antisecretory gastric acidctivity of Plectranthus barbatus previously reported (Fischmant al., 1991; Lapa et al., 1992). Plectrinone A, an abietane diter-enoid described in the plant leaves (Ruedi, 1986), inhibitedhe gastric H+,K+-ATPase with IC50 value about 10-fold greaterhan that of the classic proton pump inhibitor omeprazole.
A number of plant-derived compounds were reported tonhibit the gastric H+,K+-ATPase activity in vitro such as sal-ianolic acid A (Murakami et al., 1990), ellagic acid (Murakamit al., 1991), catechins (Murakami et al., 1992), flavonoids
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ig. 4. Concentration-inhibition relationship of plectrinone A on H+,K+-ATPasesolated from rabbit gastric mucosa. The symbols are mean ± S.E. of three assaysach in triplicates.
Murakami et al., 1999), the tetracyclic diterpenoid scopadul-iol, and scopadulcic acid B isolated from Scoparia dulcisScrophulariaceae) (Asano et al., 1990; Hayashi et al., 1991).ew of these constituents were correlated to their in vivo activitys presently reported.
Physiological secretion of hydrochloric acid is known tonvolve activation of His H2, muscarinic M3 and gastrinCK2 receptors on the parietal cell basolateral membrane. The
esponse to His involves an increase of 3′,5′-cyclic monophos-hate (cAMP) followed by activation of a cAMP-dependentrotein kinase that triggers the translocation and insertion of the+,K+-ATPase in the apical plasma membrane of the parietal
ell. Cholinergic agents and gastrin actions involve activation ofhospholipase C and production of inositol 1,4,5-triphosphatend diacylglycerol; the former increases the cytosolic calciumoncentration and the latter stimulates protein kinase C, leadingoth to activation of the proton pump (Urushidani and Forte,997; Yao and Forte, 2003). Thus, although many physiologicalargets may be involved in the decrease of acid secretion, onlynhibition of the H+,K+-ATPase may account for simultaneouslockade of both regulatory pathways.
The results obtained in isolated rabbit gastric glands showedhat the basal acid secretion, as well as that stimulated by His andethanechol were reduced by the fraction containing plectrinone, indicating that both cholinergic and histaminergic regula-
ory pathways of acid secretion were simultaneously inhibited.fter chemical purification the proton pump inhibitory activ-
ty of plectrinone A was increased by 10-fold irrespectively ofhe tested secretagogue. These data, therefore, confirmed thathe proton pump is the target for Plectranthus barbatus gastricction. This proton pump is a membrane bound enzyme thatatalyses H+ transport at the expense of ATP hydrolysis in the
nal pathway of acid secretion (Sachs et al., 1976; Forte et al.,980).
Plectranthus barbatus leaves are rich in diterpenoids, theajority of them are modified abietanoids (Abdel-Mogib et
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l., 2002; Ruedi, 1986; Ruedi and Eugster, 1972), but few ofhem were associated with biological activities. For instance,he diterpenoids barbatusin, cyclobutatusin and 3�-hydroxy--deoxybarbatusin were reported to present antitumoral effectn mice (Zelnik et al., 1977), whereas the tricyclic aromaticiterpene barbatusol produced hypotension and bradycardia innesthetized rats (Kelecom, 1983). Diterpenes abietanes isolatedrom the same species were related to antibacterial activity (Rabend van Staden, 1998). More recently, essential oils obtainedrom Plectranthus barbatus were shown to induce smooth mus-le relaxant activity involving Ca2+ influx in isolated guinea-pigreparations (Camara et al., 2003). None of the reported plantonstituents, however, was associated with the antisecretoryction presently demonstrated for plectrinone A. This diter-enoid is a highly hydroxylated compound with adjacent (ortho)ydroxyl phenolic groups at C-11 and C-12, a strong intermolec-lar hydrogen bonding between C O (C-7) and hydroxyl groupst C-14 (δ 13.56). Structure-activity relationship studies havehown that the number of hydroxyl groups and the hydroxyla-ion pattern of phenolic compounds are determinant for effectivenhibition of the H+,K+-ATPase activity (Murakami et al., 1992;
urakami et al., 1999), which may also explain the effect oflectrinone A.
Currently used proton pump inhibitors like the substitutedenzimidazoles, omeprazole and analogues, convert to theirctive metabolites in acidic compartments, and bind to essentialysteines on the luminal surface of the gastric H+,K+-ATPaseLindberg et al., 1986). A second type of proton pump inhibitors an imidazol [1,2 �] pyridine derivative, SCH 28080A, reportedo competitively bind to the K+ high affinity site of H+,K+-TPase (Wallmark et al., 1987). Preliminary kinetic analysis
ndicate a different mechanism of enzyme inhibition for plectri-one A, which is currently being investigated.
. Conclusion
Confirming the reputed antisecretory and antiulcer activitiesf Plectranthus barbatus, this study demonstrated that plec-rinone A, a known abietane diterpene of the plant leaves,nhibits the gastric H+,K+-ATPase activity. The high compli-nce to the plant extract in folk medicine to treat gastric ulcersnd hyper secretion of gastric acid is justified by the presentedndings. These data provide scientific support to the reputedfficacy of Plectranthus barbatus favoring the use of plectri-one A as the chemical marker compound for the plant extracttandardization.
cknowledgments
The authors acknowledge the pharmacognostic expertisend valuable suggestions of Prof. F.J.A. Matos and Dr. M.I.L.achado. Thanks are due to Dr. R.O. Godinho for assistance in
he H,K-ATPase extraction procedure, and to Prof. M. Yoshida
or reading the manuscript. The technical assistance of W.S.C.elisbino, M.C. Goncalo, C.M. Dores and F.R. Santos are alsocknowledged. This work received financial support from Con-elho Nacional de Desenvolvimento Cientıfico e Tecnologico
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harmacology 111 (2007) 1–7
CNPq) and Coordenacao de Aperfeicoamento de Pessoal deıvel Superior (CAPES).
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